TN-1139 APPLICATIONS HPLC-UV Analysis of Gingerol in Root Method Status: Scientifically Valid per cGMPs for Dietary Supplements

Phenomenex: Zeshan Aqeel, J Preston, Jeff Layne ChromaDex: Steve Baugh

Introduction Identity testing of raw materials for potency and verification of label nology 5 µm C18 50 x 4.6 mm were from Phenomenex, Torrance, claim data in nutraceutical formulations are requirements of dietary CA. All chromatographic conditions are specified on their corre- supplement cGMPs. Manufacturers and contract testing labs alike sponding chromatograms in Figures 1 through 7. are looking for accurate and scientifically valid methods that are The ginger root biomass reference material, ginger extract ref- suitable for use with different formulations. The complex nature of erence material, 6-Gingerol, 8-Gingerol, 10-Gingerol, 6-, nutraceuticals and botanicals often requires long analysis and dif- 9-Shogaol, 10-Shogaol, and the analytical method on ficult sample cleanup steps to resolve matrix interferences. the Luna HST C18 were provided by ChromaDex®. The formu- There were two primary goals of this project: 1) to optimize our lated products were purchased from a local health foods store. pre-exisiting method to reduce total analysis time using newer All materials were dissolved in methanol/water (80:20) with 0.1 % high efficiency HPLC technologies and 2) evaluate the quantita- TFA added as a stabilizer. Before injection, samples were filtered tive results obtained by using newly available gingerol and shogaol through a 0.45 µm Phenex™ PTFE syringe filter. reference standards versus a traditional quantitation method that relied on using capsaicin as a reference standard. Figure 1. Analytical Reference Standards Using Luna HST 2.5 µm Column Botanical Information Ginger root Column: Luna 2.5 µm C18(2) HST 20798 Part No.: 00D-4446-B0 Botanical Name Dimensions: 100 x 2.0 mm Zingiber officinale Mobile Phase: A: Water mAU B: Acetonitrile 4 7 Gradient: A/B (60/40) for 1 min to Common Names 6 (20/80) @ 9 min to (5/95) @ 100 3 5 Ginger, Jiang, Ardraka, Shunthi 2 10 min and hold 3 min Flow Rate: 0.5 mL/min Plant Description 80 Temperature: 50 °C 1 Detection: UV @ 282 nm Ginger originates from India and is now cultivated in many tropi- Sample: 1. 6-Gingerol cal countries. It is a perennial reed-like plant with leafy stems and 60 2. Capsaicin grows to 3-4 feet tall. Producing clusters of white and pink flower 3. 8-Gingerol 40 4. 6-Shogaol buds that blossom into yellow flowers, it is often used as landscap- 5. 10-Gingerol ing in subtropical homes. 6. 8-Shogaol 20 7. 10-Shogaol

Therapeutic Use Overview App ID 20798 0 The ginger is used as a spice in many cuisines in food, beverages and confections. It is also an important component of 0 2 4 6 8 10 12 14 15 min the traditional medicines of India, China, Indonesia and Japan. It has been used for motion sickness, nausea, indigestion and os- 20800 teoarthritis. The gingerol compounds it contains are well known as Figure 2. Analytical Reference Standards using Kinetex 5 µm Core-Shell Technology antioxidants. Column

mAU 4 Column: Kinetex 5 µm C18 Part No.: 00B-4601-E0 Dimensions: 50 x 4.6 mm 2 6 3 7 Mobile Phase: A: Water 5 B: Acetonitrile 40 1 Gradient: A/B (65/35) for 0.5 min to (5/95) at 5 min and hold 2 min Flow Rate: 2 mL/min Temperature: 30 °C 30 Detection: UV @ 282 nm Sample: 1. 6-Gingerol 2. Capsaicin 3. 8-Gingerol 20 4. 6-Shogaol Experimental 5. 10-Gingerol ® 6. 8-Shogaol HPLC analysis was performed using an Agilent 1100 LC System 7. 10-Shogaol (Agilent Technologies Inc., Palo Alto, CA, USA). The system was 10 optimized in order to reduce dead volume and improve perfor- • 53 % Faster Analysis Time • Equivalent Resolution mance including increasing the UV scan rate, changing the injec- • Much simpler mobile phase tor needle seat, re-plumbing the system with red PEEKsil™ Tubing (SGE), and using a semi-micro flow cell. The fully porous Luna® 0 App ID 20800 ® 2.5 µm HST C18 100 x 2.0 mm and the Kinetex Core-Shell Tech- 0 2 4 6 8 min

For additional technical notes, visit www.phenomenex.com and www.chromadex.com Page 1 of 4 TN-1139 APPLICATIONS Figure 3. Table 1. Ginger Root Biomass Sample Using Luna® HST 2.5 µm Column Calibration Curve from Kinetex 5 µm Core-Shell Technology Column Based on ChromaDex Reference Materials Column: Luna 2.5 µm C18(2) HST Compound Linearity Equation R2 LOQ Part No.: 00D-4446-B0 Dimensions: 100 x 2.0 mm 6-Gingerol Y = 0.7255x – 0.8877 0.9999 1.66 µg/mL mAU Mobile Phase: A: Water 8-Gingerol Y = 0.7403x – 0.5055 0.9999 1.66 µg/mL B: Acetonitrile 6-Shogaol Y = 0.8887x – 0.8620 0.9999 1.66 µg/mL Gradient: A/B (60/40) for 1 min to (20/80) at 1 9 min to (5/95) at 10 min and 10-Gingerol Y = 0. 5122x – 0.3766 0.9999 1.66 µg/mL 140 hold 3 min 8-Shogaol Y = 0.7898x – 0.3010 0.9999 1.66 µg/mL Flow Rate: 0.5 mL/min 10-Shogaol Y = 0.7822x – 1.4783 0.9999 1.66 µg/mL 120 Temperature: 50 °C Detection: UV @ 282 nm Sample: 1. 6-Gingerol Table 2. 100 3 2. 8-Gingerol Comparison of Method Accuracy at 500 µg/mL Using Individual Standards 3. 6-Shogaol vs. Capsaicin Reference 80 4. 10-Gingerol 5. 8-Shogaol Compound ChromaDex Standards Capsaicin 4 6. 10-Shogaol 6-Gingerol 100.0 % 83.8 % 60 8-Gingerol 100.0 % 82.0 % 6 6-Shogaol 100.0 % 68.4 % 40 2 10-Gingerol 100.0 % 118.6 % 8-Shogaol 100.0 % 76.9 % 5 20 App ID 20799 10-Shogaol 100.0 % 77.8 % Accuracy based on three replicated injections of the of 500 µ/mL standard 0

0 2 4 6 8 10 12 14 min Table 3. Comparison of Quantitative Values from a Ginger Root Biomass Sample

Figure 4. Gingerol Extract (µg/g) Gingerol Biomass (µg/g) Ginger Root Biomass Sample Using Kinetex® 5 µm Core-Shell Technology Compound Reference Standards Capsaicin Reference Standards Capsaicin Column20802 6-Gingerol 232 280 866 801 8-Gingerol 70 46 255 136 mAU Column: Kinetex 5 µm C18 • Equivalent performance Part No.: 00B-4601-E0 6-Shogaol 125 118 629 495 even in plant extracts Dimensions: 50 x 4.6 mm 10-Gingerol 74 68 251 192 100 Mobile Phase: A: Water B: Acetonitrile 8-Shogaol 16 16 91 78 1 Gradient: A/B (65/35) for 0.5 min to (5/95) 10-Shogaol 43 45 177 161 at 5 min and hold 2 min Total 583 573 2,270 1,863 80 Flow Rate: 2 mL/min ChromaDex reference standards allow for more accurate determination of gingerol content, especially Temperature: 30 °C in difficult formulations Detection: UV @ 282 nm Sample: 1. 6-Gingerol Figure 6. 60 2. 8-Gingerol 3. 6-Shogaol Analysis of Liquid Filled Gel Cap Formulation Using Kinetex 5 µm Core- 4. 10-Gingerol Shell20803 Technology Column 5. 8-Shogaol 3 6. 10-Shogaol mAU Column: Kinetex 5 µm C18 40 Part No.: 00B-4601-E0 800 Dimensions: 50 x 4.6 mm 4 Mobile Phase: A: Water B: Acetonitrile 20 2 6 Gradient: A/B (65/35) for 0.5 min to (5/95)

ID 20799 at 5 min and hold 2 min

5 p p 600 Flow Rate: 2 mL/min A

1 Temperature: 30 °C 0 Detection: UV @ 282 nm

0 1 2 3 4 5 6 min

400

Figure 5. Representative Calibration Curve for Gingerols from 1.66 µg/g to 500 µg/mL 3

200 8-Gingerol 4

 2 6 5 App ID 20803 0

0 2 4 6 8 min Response Peak Compound Conc./Capsule (mg)

 1 6-Gingerol 15.08  0 100 200 300 400 500 600 2 8-Gingerol 3.13 Concentration 3 6-Shogaol 5.84 4 10-Gingerol 5.16 5 8-Shogaol 0.8 6 10-Shogaol 2.03 Total 32.04 Label Claim 30

Page 2 of 4 For additional technical notes, visit www.phenomenex.com and www.chromadex.com TN-1139 APPLICATIONS Figure 7. method. Methods were shown to be linear over a range of 5 to Analysis of Powder Filled Gel Cap Formulations Using Kinetex® 5 µm Core- 2,000 µg/mL. The LOQ was determined to be 1.66 µg/mL for all Shell20804 Technology Column of the compounds. This methodology produced a Signal to Noise mAU ratio of > 10 for the 10-Gingerol peak, which was the smallest Column: Kinetex 5 µm C18 peak of interest from the isolated standard (Table 1). Part No.: 00B-4601-E0 Dimensions: 50 x 4.6 mm Mobile Phase: A: Water There are several different ways to quantitate the components of B: Acetonitrile interest with this methodology. One way is to produce a linearity Gradient: A/B (65/35) for 0.5 min to (5/95) curve for each component based on the ChromaDex reference at 5 min and hold 2 min Flow Rate: 2 mL/min standard and use the corresponding linear equation from this 1 Temperature: 30 °C data for each component. Another is to spike each sample with Detection: UV @ 282 nm 10 a known amount of capsaicin and quantitate against this internal standard, which has been done historically because there were no suitable reference materials available.

3 The quantitation based on individual reference standards provided 4 2 higher accuracy than the capsaicin method (Table 2). When this 6 5 quantitation was applied to real samples, the calculated concen-

App ID 20804 tration in the ginger extract gave higher calculated values for total 0 gingerol (Table 3). When calibrating for compounds in a difficult

0 1 2 3 4 5 min matrix, it is always preferred to use a reference standard of the Peaks Compound Conc./Capsule (mg) actual compound of interest. When using alternative compounds, 1 6-Gingerol 0.144 there is always the potential for unknown co-elutions that could 2 8-Gingerol 0.026 bias the data high or low. 3 6-Shogaol 0.05 4 10-Gingerol 0.055 The final experiment was to analyze commercially available formu- 5 8-Shogaol 0.012 lations and determine if the results were similar to the label claim. 6 10-Shogaol 0.025 To ensure that we properly tested our new assay, we attempted Total 0.399 to choose difficult formulations such as those with multiple active Label Claim ??? components and those with gel cap pills. Analysis of two formu- lated products showed that the values obtained are consistent Results and Discussion with label claims. The original method supplied by ChromaDex was already opti- ® mized on a high efficiency Luna HST 2.5 µm fully porous column Conclusion and is much faster than similar published separations. The meth- HPLC analysis times were reduced by over 50 % by adapting odology has been in routine use by ChromaDex for contract ana- the current method from the Luna HST to the Kinetex Core-Shell lytical testing for the past 5 years and has been shown to provide Technology method. We performed experiments to verify that the good separation and accurate results for gingerol components in methodology was suitable for nutraceutical products. Final results a variety of botanical products. indicate that this method is robust and ready for routine sample The Kinetex Core-Shell Technology allows scientists to achieve analysis. substantially higher chromatographic efficiencies at much lower Quantitative determination using individual reference standards pressures than the equivalent fully porous material. The Kinetex was the best way to achieve accurate quantitation, especially in materials enhance the performance of any existing HPLC plat- formulations containing other botanical products. The label claims form, including UHPLC systems. For those labs that have older of some products are also quite difficult to interpret, making it dif- HPLC systems without high pressure capabilities, the Kinetex ficult for a consumer to understand the value of the botanical they 2.6 µm and 5 µm columns can substantially improve the useable are taking. Providing standardized reference methods for analysis lifetime of these systems. is the first step to ensuring quality in nutraceutical products. The increased separation ability of the Kinetex 5 µm column al- lowed the column length to be shortened by 67 % to a 50 mm length. This reduction in length, combined with gradient condi- tions that were scaled to match the new column dimensions re- Scienti cally Valid duced the analysis time by over 50 %. Resolution and sensitivity were similar to the existing method (Figures 1 and 2). Section 21CFR111.320 of cGMPs for Dietary Supplements requires you to “identify and use an appropriate scientifically valid method for each estab- When analyzing botanicals and nutraceuticals, the separation of lished specification for which testing or examination is required to determine whether the specification is met”. The FDA does not elaborate on what is standards can often be misleading since the plant extract can considered a scientifically valid method in the cGMPs. ChromaDex has contain many other endogenous components that could lead to defined scientifically valid as a method that meets minimum linearity, preci- poor results. To demonstrate specificity of the new method, ginger sion, sensitivity and range requirements. These requirements are outlined in root biomass reference materials were compared using the fully an FDA laboratory document, ORA LABORATORY PROCEDURE Food and Drug Administration, ORA-LAB.5.4.5. This laboratory guidance document porous Luna HST method versus the Kinetex Core-Shell Technol- defines minimal performance attributes for selected methods of analysis ogy method. Resolution from matrix components was similar on and has been applied by ChromaDex to the selection of methods that are both columns (Figures 3 and 4). fit for purpose in the dietary supplements industry. According to the above definition, the method detailed in this document is considered scientifically Having demonstrated that the new method provided equivalent valid as application to the cGMP requirements. Product specific, full meth- od validations according to AOAC guidelines can be applied to customer results, we performed experiments to determine linearity, accu- samples upon request, to further document method performance in specific racy, range, and limit of quantitation (LOQ) of the new Kinetex samples and matrices.

For additional technical notes, visit www.phenomenex.com and www.chromadex.com Page 3 of 4 TN-1139 APPLICATIONS

ChromaDex Ordering Information ChromaDex, Inc. Reference Standards 10005 Muirlands Blvd., Suite G Description Quantity Part No. 6-Gingerol (P) 5 mg ASB-00007164-005 Irvine, CA 92618 USA 6-Gingerol (P) 10 mg ASB-00007164-010 Tel: +1.949.419.0288 6-Gingerol (P) 25 mg ASB-00007164-025 Fax: +1.949.419.0294 8-Gingerol (P) 5 mg ASB-00007163-005 [email protected] 8-Gingerol (P) 10 mg ASB-00007163-010 www.chromadex.com 8-Gingerol (P) 25 mg ASB-00007163-025 Please see www.chromadex.com for a 10-Gingerol (P) 5 mg ASB-00007162-005 listing of international distributors 10-Gingerol (P) 10 mg ASB-00007162-010 10-Gingerol (P) 25 mg ASB-00007162-025

6-Shogaol (P) 5 mg ASB-00019211-005 6-Shogaol (P) 10 mg ASB-00019211-010 6-Shogaol (P) 25 mg ASB-00019211-025

8-Shogaol (P) 5 mg ASB-00019212-005 8-Shogaol (P) 10 mg ASB-00019212-010 Australia Luxembourg 8-Shogaol (P) 25 mg ASB-00019212-025 t: 02-9428-6444 t: +31 (0)30-2418700 f: 02-9428-6445 f: +31 (0)30-2383749 [email protected] [email protected] 10-Shogaol (P) 5 mg ASB-00019214-005 10-Shogaol (P) 10 mg ASB-00019214-010 Austria Mexico 10-Shogaol (P) 25 mg ASB-00019214-025 t: 01-319-1301 t: 001-800-844-5226 f: 01-319-1300 f: 001-310-328-7768 [email protected] [email protected] Capsaicin(methyl-n-vanillyl-6-Noneneamide)(P) 5 mg ASB-00003135-005 Capsaicin(methyl-n-vanillyl-6-Noneneamide)(P) 10 mg ASB-00003135-010 Belgium The Netherlands Capsaicin(methyl-n-vanillyl-6-Noneneamide)(P) 25 mg ASB-00003135-025 t: 02 503 4015 (French) t: 030-2418700 t: 02 511 8666 (Dutch) f: 030-2383749 f: +31 (0)30-2383749 [email protected] Ginger Standards Kit (P) 7x 5 mg KIT-00007615-005 [email protected] New Zealand Ginger Standards Kit (P) 7x 10 mg KIT-00007615-010 t: 09-4780951 Canada Botanical Reference Materials t: (800) 543-3681 f: 09-4780952 f: (310) 328-7768 [email protected] Description Quantity Part No. [email protected] Norway Ginger (Zingiber officinale) Root (BRM) 5 g ASB-00030290-005 Denmark t: 810 02 005 Ginger (Zingiber officinale) Root (RGBRM) 5 g ASB-00030863-005 t: 4824 8048 f: +45 4810 6265 Ginger (Zingiber officinale) Root peeled (VBRM) 5 g ASB-00030956-005 f: +45 4810 6265 [email protected] [email protected] Puerto Rico Finland t: (800) 541-HPLC Phenomenex Ordering Information t: 09 4789 0063 f: (310) 328-7768 Kinetex® Core-Shell HPLC Columns f: +45 4810 6265 [email protected] SecurityGuard™ [email protected] Sweden SecurityGuard 5 µm Columns (mm) ULTRA Cartridges* ULTRA Cartridges* France t: 08 611 6950 f: +45 4810 6265 50 x 2.1 3/pk 50 x 4.6 100 x 4.6 150 x 4.6 250 x 4.6 3/pk t: 01 30 09 21 10 XB-C18 00B-4605-AN AJ0-8782 00B-4605-E0 00D-4605-E0 00F-4605-E0 00G-4605-E0 AJ0-8768 f: 01 30 09 21 11 [email protected] [email protected] C18 00B-4601-AN AJ0-8782 00B-4601-E0 00D-4601-E0 00F-4601-E0 00G-4601-E0 AJ0-8768 United Kingdom PFP 00B-4602-AN AJ0-8787 00B-4602-E0 00D-4602-E0 00F-4602-E0 00G-4602-E0 AJ0-8773 Germany t: 01625-501367 Phenyl-Hexyl 00B-4603-AN AJ0-8788 00B-4603-E0 00D-4603-E0 00F-4603-E0 00G-4603-E0 AJ0-8774 f: 01625-501796 t: 06021-58830-0 for 2.1 mm ID for 4.6 mm ID [email protected] f: 06021-58830-11 * SecurityGuard ULTRA cartridges require holder, Part No. AJ0-9000. [email protected] United States India t: (310) 212-0555 t: 040-3012 2400 f: (310) 328-7768 f: 040-3012 2411 [email protected] [email protected] All other countries: Corporate Office USA If Phenomenex products in this technical note do not provide at least an equiva- Ireland lent separation as compared to other products of the same phase and comparable t: 01 247 5405 t: (310) 212-0555 f: +44 1625-501796 f: (310) 328-7768 dimensions, return the product with comparative data within 45 days for a FULL [email protected] [email protected] REFUND. Italy Terms and Conditions t: 051 6327511 Subject to Phenomenex Standard Terms and Conditions which may be viewed at TN97380912_W f: 051 6327555 www.phenomenex.com/TermsAndConditions. [email protected] Trademarks www.phenomenex.com Luna and Kinetex are registered trademarks of Phenomenex. Phenex is a trademark of Phenomenex. Agilent 1100 is a registered trademark of Agilent Technologies, Inc. ChromaDex is a registered trademark of ChromaDex Corp. PEEKsil Phenomenex products are available worldwide. For the distributor in is a trademark of SGE your country, contact Phenomenex USA, International Department at [email protected] © 2012 Phenomenex, Inc. All rights reserved.

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